High-affinity binding at quadruplex–duplex junctions: rather the rule than the exception

Abstract Quadruplex-duplex (Q–D) junctions constitute unique structural motifs in genomic sequences. Through comprehensive calorimetric as well as high-resolution NMR structural studies, Q–D junctions with a hairpin-type snapback loop coaxially stacked onto an outer G-tetrad were identified to be most effective binding sites for various polycyclic quadruplex ligands. The Q–D interface is readily recognized by intercalation of the ligand aromatic core structure between G-tetrad and the neighboring base pair. Based on the thermodynamic and structural data, guidelines for the design of ligands with enhanced selectivity towards a Q–D interface emerge. Whereas intercalation at Q–D junctions mostly outcompete stacking at the quadruplex free outer tetrad or intercalation between duplex base pairs to varying degrees, ligand side chains considerably contribute to the selectivity for a Q–D target over other binding sites. In contrast to common perceptions, an appended side chain that additionally interacts within the duplex minor groove may confer only poor selectivity. Rather, the Q–D selectivity is suggested to benefit from an extension of the side chain towards the exposed part of the G-tetrad at the junction. The presented results will support the design of selective high-affinity binding ligands for targeting Q–D interfaces in medicinal but also technological applications.


NMR experiments
A WATERGATE W5 pulse scheme was employed for water suppression in one-dimensional (1D) and twodimensional (2D) NOESY experiments on a 90% H2O/10% D2O phosphate buffer solution. For DQF-COSY, TOCSY, and 1 H-13 C HSQC experiments, a 3-9-19 W3 binomial-type sequence was used for solvent suppression. 1 H-13 C HSQC spectra were recorded with 4K x 500 data points, a 1 s recycle delay, and 7500 Hz in the F1 dimension to include aromatic C8/C6/C2 resonances of the nucleobases. TOCSY spectra with a DIPSI-2 isotropic mixing scheme and an 80 ms mixing time as well as DQF-COSY spectra were recorded with 4K x 500 data points. 2D NOESY (80, 150, and 300 ms mixing time) and EASY-ROESY spectra (80 ms mixing time, 50° spinlock angle) were acquired with 2K x 1K data points. For all 2D homonuclear experiments a 2 s recycle delay was used. Data were zero-filled to 4K x 1K data points and processed with squared sine-bell window functions except for time domain data of 1D experiments that were multiplied with an exponential function.

Restraints for structure calculations
For the structure calculations, distance restraints were assigned based on the crosspeak intensities in NOESY spectra. For non-exchangeable protons and intramolecular ligand contacts, these were set according to 2.9 ± 1.1 Å for strong crosspeaks, 4.0 ± 1.2 Å for crosspeaks of medium intensity, 5.5 ± 1.5 Å for weak crosspeaks, 6.0 ± 1.5 Å for very weak crosspeaks, and 5.0 ± 2.0 Å for ambiguous crosspeaks due to signal overlap. For labile protons, distances were restrained according to 2.9 ± 1.1 Å for strong crosspeaks, 4.0 ± 1.2 Å for crosspeaks of medium intensity, 5.0 ± 1.2 Å for weak crosspeaks, 6.0 ± 1.2 Å for very weak crosspeaks, and 5.0 ± 2.0 Å for ambiguous crosspeaks due to signal overlap. Intermolecular ligand-DNA contacts were set to 4.0 +1.5/-2.0 Å for strong crosspeaks, 5.5 +1.5/-2.0 Å for weak crosspeaks, 6.0 +1.5/-2.0 Å for very weak crosspeaks, and 5.0 ± 2.0 Å for ambiguous crosspeaks due to signal overlap. Torsion angles χ were either set to anti (170º -310º) or syn (25º -95º). Sugar pucker information was derived from DQF-COSY crossspeaks, whose in-phase/antiphase pattern is determined by corresponding coupling constants and enables differentiation between a north (pseudorotation angle of 0º -36º) and south sugar pucker (pseudorotation angle of 144º -180º) through the Karplus-relationship. Distance restraints for hydrogen bonds were added for the G-tetrads and all Watson-Crick base pairs and additional planarity restraints were added to the quadruplex core. Force constants for in vacuo simulations were set to 40 and 50 kcal·mol -1 ·Å -2 for NOE-based distance and hydrogen bond restraints, 200 kcal·mol -1 ·rad -2 for glycosidic torsion angle and sugar pucker restraints, and 30 kcal·mol -1 ·Å -2 for G-tetrad planarity restraints. Additional chirality restraints with a restraint energy of 10 kcal·mol -1 ·rad -2 were added for calculations of the QD3-sbl -Phen-DC3 complex. For subsequent simulations in explicit water, only NOE-based distance and hydrogen bond restraints with restraint energies of 15 and 25 kcal·mol -1 ·Å -2 were employed.  a Average values with root-mean-square deviations obtained from three independent measurements in 20 mM potassium phosphate buffer, pH 7.0, 100 mM KCl, and 5% DMSO at 40 ºC; data were fitted with one or two sets of binding sites with fit parameters of a second lower affinity binding site shown on a grey background. b -TΔSº = ΔGº -ΔHº with ΔGº = -RTlnKa.
S7 Figure S1. NMR imino proton spectral region of QD2-l-2bp. In addition to imino resonances of the quadruplex G-core, a GC Watson-Crick imino signal is also observed. Another potential Watson-Crick imino proton resonance seems to be broadened beyond detection through increased flexibility and associated fast solvent exchange under the present conditions. The spectrum was acquired with a strand concentration of 0.3 mM in 120 mM K + buffer, pH 7.0, at 40 o C. Note: Resonance assignments and the determination of a three-dimensional NMR structure of the same sequence has been published recently under different conditions (20 °C, 10 mM potassium phosphate buffer, pH 7.0; PDB 7PNE; BMRB 34664). 1 The present NMR analysis demonstrates that different buffer conditions as used here did result in some minor chemical shift changes but did not impact the formed topology.

Resonance assignments of a QD3-sbl complex with Phen-DC3
Continuous sugar-base NOE contacts can be followed from T1 through G5 and up to syn-G36 as well as along the other three G-columns with interruptions by the propeller-type loops ( Figure S7B,C). The second and the third G-column can be differentiated by NOE contacts of A11 in the second propeller loop, showing sequential contacts to G12 but also additional contacts in particular through its H2 proton to G9 H1'. Sequential NOEs also connect all residues from C19 to G35 of the duplex stem. However, in contrast to the free QD3-sbl, NOE connectivities from the 3'-tetrad to the duplex are interrupted. The presence of G36 adopting a syn conformation is demonstrated by its strong H8-H1' intra-nucleotide crosspeak and its typical downfield-shifted G 13 C8 resonance observed in a 1 H-13 C HSQC experiment ( Figure S7A). Stereospecific assignments of the H2'/H2" sugar protons are based on H1'-H2' crosspeak intensities in NOESY experiments acquired at short mixing times ( Figure S8A). Sugar puckers were grouped into south-type and north-type conformations depending on the pattern and intensity of DQF-COSY H1'-H2' and H1'-H2" crosspeaks ( Figure S8B). Most of the residues were found to be in a south conformation with some sugars remaining ambiguous and left unassigned. In contrast, T1 and G36 could be restrained to a north sugar conformation. Guanine imino (H1) protons of the G-core were assigned without specific isotope labeling by following the characteristic pattern of intra-and inter-tetrad H8-H1 contacts. Their assignment was confirmed by imino-imino connectivities typical for an all-homopolar stack of G-tetrads with polarities of the three tetrads when going from hydrogen bond donor to acceptor along G4→G7→G12→G16, G5→G8→G13→G17, and G36→G9→G14→G18 ( Figure   S9D,E). Contacts from H1' sugar protons of 5'-overhang residues to four imino resonances in the 5'-tetrad further confirmed their assignment ( Figure S9F). Overall, the topology of QD3-sbl was retained after the addition of 1 equivalent Phen-DC3. Unlike free QD3-sbl, a prominent crosspeak correlating G35 H8 with G36 H1 is missing in the complex ( Figure S9E) The two most downfield-shifted isochronous H4 amide protons at about 11.8 ppm serve as a convenient starting point by exhibiting NOE contacts to the quinoline moiety including N-methyl protons ( Figure S9A).
Various contacts of phenanthroline protons H1, H2, and H3 but also of the H4 amide and quinoline protons closest to the phenanthroline moiety with residues G18, C19, G35, and G36 point to their location at the Q-D junction ( Figure S7 and S9). On the other hand, quinoline protons distant from the phenanthroline ring system exhibit NOE contacts to the exposed interfacial G-tetrad residues G9 and G14. These connectivities suggest S14 the phenanthroline to be sandwiched between base pair and G-tetrad at the Q-D junction whereas both Chemical structure of Phen-DC3 with atom numbering as used in this study. S15 Figure S7. NMR spectra with assignments for a 1:1 complex of QD3-sbl (0.5 mM) with Phen-DC3 acquired at 30 °C in 10 mM potassium phosphate buffer, pH 7. (A) 1 H-13 C HSQC spectral region with H6/H8(ω2)-C6/C8 (ω1) correlations; the crosspeak with downfield-shifted 13 C8 of syn-G36 is labeled in red; a G30 H8-C8 correlation only observed at lower threshold levels is marked by a cross. (B) H6/H8(ω2)-H3'(ω1) and (C) H6/H8(ω2)-H1'(ω1) spectral region of a NOESY spectrum (300 ms mixing time). NOE sequential connectivities for the quadruplex and the duplex domain are traced by black and blue lines, respectively. Syn-G36 with its strong intra-nucleotide H8-H1' crosspeak shows a weak NOE contact of its H1' proton to G5 H8 across the anti-syn step with antiparallel strand orientation (highlighted by the red rectangular pattern). Intra-and intermolecular ligand contacts are labeled with the ligand proton written in blue.  NOESY spectra were acquired at 30 °C in 10 mM potassium phosphate buffer, pH 7, with a 300 ms mixing time except for the spectral region in (H) that derives from a NOESY experiment with an 80 ms mixing time.

Resonance assignments of free Q3-sbl2 and of its complex with Phen-DC3
Non-interrupted base-sugar NOEs can be traced from T1 to G5 and syn-G22, demonstrating a first truncated G-column with a broken G-tract and the open position filled by the 3'-terminal G22 (S11C). In analogy to QD3sbl, second and third G-columns can be distinguished by following contacts of A11 in the second propeller loop. While a continuous NOE walk can be traced from G16 to T19, a long-range contact between G20 and G18 identifies the fourth G-column and the TGT lateral snapback loop. The presence of a single syn-guanosine at position 22 is additionally corroborated by its typical 13 C8 chemical shift in 1 H-13 C HSQC spectra ( Figure   S11B). Based on a parallel quadruplex, imino protons were unambiguously assigned without specific isotope labeling by intra-and inter-tetrad H8-imino connectivities and further confirmed by their sequential imino-imino contacts ( Figure S11D,E). Also, a NOE crosspeak between the G22 imino and the T21 H6 proton demonstrates positioning of the snapback loop above the 3'-tetrad, effectively protecting an observable G22 amino proton from solvent exchange.
Assignments for the quadruplex in the 1:1 complex with Phen-DC3 closely follows the assignments of the free Q3-sbl2, demonstrating a conserved parallel topology, a lateral snapback loop, homopolar tetrad stacking, and a G22 amino proton protected from solvent exchange ( Figure S12). Various contacts from Phen-DC3 quinoline protons to the quadruplex can be observed, including contacts to all G imino protons in the 5'-tetrad but also to T1 H1' in the 5'-overhang. These unambiguously show that the Phen-DC3 ligand stacks onto the 5'-face of the quadruplex ( Figure S12C-F). However, some unidentified crosspeaks to protons of the 3'-tetrad also suggest small amounts of a minor complex with a putative Phen-DC3 binding at the 3'-tetrad ( Figure   S12D). Interestingly, in contrast to Phen-DC3 intercalated at the Q-D junction of QD3-sbl, symmetry-related ligand protons are subject to chemical exchange through a flip of the ligand as shown by exchange crosspeak in a ROESY experiment ( Figure S13).     Resonance assignments of free QD2-l and of its complex with PIQ-4m The imino proton spectral region of the QD2-l hybrid shows six and eight imino resonances with chemical shifts typical of Watson-Crick base pairs and of more upfield shifted G iminos involved in Hoogsteen hydrogen bonds, respectively. Identifying four syn-anti steps along the G-columns, a two-layered antiparallel G-quadruplex can be established. The first and third G-columns were assigned based on continuous sugar-base NOE contacts to the following TT lateral loops. The second G-column is identified due to non-interrupted NOE connectivities from syn-G5 along the duplex stem loop up to G21 ( Figure S14C,D). Various contacts at the Q-D interface including G21 H8 to G22 H1 and C7 H4 to G6 H1 positions the duplex stem loop coaxially with the G-core. A typical heteropolar stacking pattern can be observed through strong non-sequential imino-imino contacts such as between G26 H1 and G22 H1. G-tetrad polarity following hydrogen bond donor to acceptor runs along G1→G6→G22→G27 and G2→G26→G23→G5. Intra-tetrad NOE contacts between G imino and G H8 protons of either syn-or anti-Gs determine the quadruplex groove width. Thus, the two TT lateral loops bridge a narrow groove while the duplex stem loop bridge the wide groove of the quadruplex.
For the 1:1 complex of QD2-l with the PIQ-4m ligand, similar sugar-base NOE connectivities as found for the free hybrid identify the first and third G-column followed by the two TT lateral loops and the duplex hairpin loop with uninterrupted NOE connectivities from C7 to G21. However, interruption of sequential contacts at the junction from G6 to C7 indicates ligand intercalation. Syn-guanines were assigned by their downfield-shifted 13 C8 resonance while three adenine H2 resonances were identified by their H2-C2 correlations in a 1 H-13 C HSQC spectrum ( Figure S16C). Stereospecific assignments of H2'/H2" protons were based on a NOESY experiment with short mixing times (80 ms) and the following determination of sugar conformations made use of the pattern and intensity of H1'-H2' and H1'-H2" crosspeaks in a DQF-COSY spectrum ( Figure S17). Except for T3 and T24, located in lateral loops bridging the narrow groove, all assigned sugar puckers are in the south domain of the pseudorotational cycle. Imino protons were assigned by following exchange crosspeaks between the free and complexed QD2-l hybrid ( Figure S15) and heteropolar tetrad stacking was confirmed by characteristic intra-and inter-tetrad H8-imino NOE contacts. Taken together, the two-layered antiparallel topology of free QD2-l with exclusive syn-anti steps along the G-columns, two TT lateral loops bridging a narrow groove, and one duplex stem loop bridging a wide groove was retained after ligand addition. However, sequential contacts bridging the quadruplex-duplex interface were lost.
Ligand proton resonances were assigned by a combination of COSY and NOESY experiments. Spin systems with corresponding COSY correlations derive from protons in the phenyl and the fused indole and quinoline ring systems ( Figure S18F). Discrimination of quinoline and indole resonances was enabled by a strong NOE crosspeak from a phenyl proton to quinoline H1 and H2 protons. Additional NOE crosspeaks of methyl substituents of the quinoline and indole moiety were observed to ring protons in their proximity, with the quinoline N-methyl proton resonating close to the water signal ( Figure S18D,E). A strong NOE contact connects the indole NH with a resonance at about 8 ppm ( Figure S18G). TOCSY and ROESY experiments identified the latter as being two isochronous ortho-positioned phenyl protons, explaining the observation of only a single scalar coupled proton pair of the phenyl ring in a DQF-COSY spectrum. The NH16 amide is assigned by following NOE contacts from adjacent phenyl protons with connectivities continuing to the aliphatic S27 side chain (Figures S18F, S19A). Methylene protons H17 adjacent to the amide were found to be nonequivalent, indicating a restricted C-C bond rotation upon binding. Other resonances of the aliphatic side chain were identified through their mutual scalar couplings observed in COSY and TOCSY experiments ( Figure   S19B). It should be noted that NOE crosspeaks for aliphatic side chain protons are rather weak and broadened due to a high flexibility towards the terminus with changing signs for NOE crosspeaks of terminal ethyl protons (not shown).